Method and kit for determining the probability that a patient will develop a severe case of dengue

Information

  • Patent Grant
  • 9933426
  • Patent Number
    9,933,426
  • Date Filed
    Thursday, February 12, 2015
    9 years ago
  • Date Issued
    Tuesday, April 3, 2018
    6 years ago
Abstract
A method for determining, in vitro, the probability of a patient developing severe dengue, based on a blood sample, according to a) the quantity in the blood sample of at least one marker, which is olfactomedin 4, is determined, b) the quantity of olfactomedin 4 determined in step a) is compared with a reference quantity of the marker obtained from a group of individuals who have been diagnosed with non-severe dengue, wherein, if the quantity of olfactomedin 4 determined in step a) is greater than the reference quantity established in step b), it is determined that the patient will develop severe dengue.
Description

The subject of the present invention is a method for the early prediction of severe dengue or hemorrhagic dengue using protein markers.


Over the past 30 years, dengue, a viral disease transmitted by urban hematophagous mosquitoes of the Aedes genus has worryingly spread throughout the world. It is currently a real public health problem for more than one hundred countries located in the subtropical zone, particularly in the Pacific West, South America and South-East Asia zones. The emergence of the disease is largely due to the population explosion and to chaotic urbanization. Climatic abnormalities also play a not insignificant role.


In this respect, dengue could emerge in the western regions of the world which until now have been spared the virus. Thus, Aedes albopictus, one of the vectors of the disease, has recently been found in the North of Italy and in the South of France. Most recently, autochthonous cases of dengue have been recorded in the South of France. It is estimated that close to three billion people are exposed to the risks of dengue. Close to one million hospitalizations are registered yearly and there have been thousands of deaths. Children are the main victims of the disease.


The dengue virus is a single-stranded, positive-polarity enveloped RNA virus of the family Flaviviridae. The genome of the virus (11 000 nucleotides) encodes a polyprotein of approximately 3400 amino acids which undergoes co- and post-translational cleavage which results in structural proteins (C, prM, E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). There are 4 viral serotypes (DV1 to DV4), which can coexist in endemic zones. There is approximately 70% sequence homology between the various serotypes. Infection by a given serotype confers long-term immunity for this serotype. Cross-protection lasts only a few months: reinfection is therefore possible with a different serotype.


Infection begins with a bite from a mosquito infected with one of the dengue viruses. Incubation, the period during which the virus replicates in the blood without however giving rise to any symptoms, generally lasts from 4 to 10 days. The first signs occur after the incubation period.


In its conventional form (“conventional” dengue fever: DF), dengue is characterized by sudden-onset hyperthermia accompanied by one or more of the following symptoms: shivering, headaches, joint and/or muscle pain, nausea and vomiting. A rash may also appear, generally on around the 5th day of symptoms. This acute febrile stage, which corresponds to the viremic phase, generally lasts from 3 to 5 days (extremes: 2 to 7 days). More than 95% of cases will have no signs of severe illness and will recover with no complications in under 7 days.


In 2 to 4% of cases, the patient may develop a critical phase characterized by a more or less severe plasma leakage syndrome and an increased hematocrit level, leading to dengue hemorrhagic fever: DHF. This phase typically (but not necessarily) appears at the time of defervescence, around the 4th or 5th day. It is generally brief (24 to h) but may develop into a severe form characterized by major hemorrhagic manifestations, a state of shock and/or the failure of one or more organs. Development into a severe form is most often signalled by one (or more) warning sign(s), such as:

    • fever (temperature of greater than 39° C.) after the 5th day;
    • intense abdominal pain, persistent diarrhea, uncontrollable vomiting and complete refusal of food;
    • edemas and/or minor effusion;
    • bleeding of mucous membranes which does not stop automatically;
    • pronounced lethargy or restlessness;
    • thrombocytopenia;
    • signs of hemoconcentration.


In the most severe cases, the leaking of plasma can lead to deadly hypovolemic shock (Dengue Shock Syndrome: DSS) if the patient is not rapidly treated. Rare but deadly hepatic and neurological involvement is also associated with the severity of the disease. The mortality rate, which is variable according to epidemics, can reach 5% of established DHF cases. This rate can increase up to 20% without hospital care or appropriate treatments.


To simplify, these severe cases will be referred to as severe dengue in the remainder of the description, as opposed to conventional dengue, DF.


90% of cases of severe dengue occur during secondary infection with a heterologous serotype, and 10% during primary infection, generally in infants aged from 6 months to 1 year. There are several factors which influence the severity of the infection, such as the factors of the host, serotype and genotype of the virus, the order and time between successive infecting viruses, the quality and quantity of cross-reactive antibodies and the CD4/CD8 response. Studies have shown a correlation between viral load and severity of the disease. The exact causes of the occurrence of severe dengue are, however, still not known. Up until now, no specific determining factor for virulence has been demonstrated. Furthermore, since there is no vaccine against the dengue virus, the only treatments available are symptomatic treatments. Consequently, it is important to be able to monitor epidemics and to predict severe cases for appropriate hospital care.


The methods currently used to diagnose dengue do not make it possible to predict the development of severe dengue. At the very most, serological methods make it possible to distinguish between primary and secondary infections and molecular methods make it possible to detect the virus and to carry out serotyping [1, 2, 3, 4].


The present invention provides a solution to the problems presented above by means of a method which allows both early and specific detection of proteins in a blood sample making it possible to predict patients developing severe dengue. Indeed, the inventors found, surprisingly, that proteins from the host were expressed more or less abundantly (overexpressed/underexpressed) in cases of patients developing severe dengue, compared to the amount or expression thereof in cases of patients remaining with conventional dengue (that is to say not developing severe dengue) in blood samples consisting, for example, of plasma. Most particularly, they have demonstrated for the first time and completely unexpectedly that olfactomedin 4 (OLFM4) is overexpressed in the case of patients developing severe dengue and thus constitutes a marker for predicting severe dengue.


Thus, a subject of the present invention is a method for predicting, in vitro, the probability of a patient developing severe dengue, based on a blood sample, wherein:

    • a) the quantity in said blood sample of at least one marker, which is olfactomedin 4, is determined,
    • b) the quantity of olfactomedin 4 determined in step a) is compared with a reference quantity of said marker obtained from a group of individuals who have been diagnosed with non-severe dengue, wherein, if the quantity of olfactomedin 4 determined in step a) is greater than the reference quantity established in step b), it is predicted that the patient will develop severe dengue.


According to the method of the invention, it is also possible to determine, in step a), the quantity in the blood sample of at least one other marker chosen from platelet factor 4 and α2-macroglobulin or the respective quantities of the two markers and, in step b), the quantity of the marker or of the two markers of step a) is compared with a reference quantity obtained from a group of individuals who have been diagnosed with non-severe dengue and, if the quantity of platelet factor 4 and/or α2-macroglobulin determined in step a) is less than the reference quantity established in step b), it is determined that the patient will develop severe dengue.


The invention also relates to a kit for the in vitro prediction of severe dengue, comprising:

    • a binding partner for olfactomedin 4,
    • a binding partner for the dengue virus NS1 protein.


The kit may also comprise a binding partner for platelet factor 4 (PF4) and/or a binding partner for a2-macroglobulin (A2M).


Definitions

The term “blood sample” is intended to mean whole blood, serum and plasma.


The term “group of individuals who have been diagnosed with non-severe dengue”, used to determine the reference quantity of the marker of interest, is intended of course to mean that the group of individuals has not developed severe dengue. Thus, in step b) the quantity of platelet factor 4 determined in step a) is compared with a reference quantity of said marker obtained from a group of individuals who have been diagnosed with dengue without having developed severe dengue.


The term “binding partner” is intended to mean, for example, receptors, antibodies, antibody fragments, antibody analogs and any other ligand capable of binding to a protein.


The binding-partner antibodies are, for example, either polyclonal antibodies or monoclonal antibodies.


The polyclonal antibodies may be obtained by immunization of an animal with the appropriate immunogen, followed by recovery of the desired antibodies in purified form, by taking the serum of said animal, and separation of said antibodies from the other serum constituents, especially by affinity chromatography on a column to which is bound an antigen specifically recognized by the antibodies.


The monoclonal antibodies may be obtained by the hybridoma technique, the general principle of which is summarized below.


Firstly, an animal, generally a mouse, is immunized with the appropriate immunogen, and the B lymphocytes of said animal are then capable of producing antibodies against this antigen. These antibody-producing lymphocytes are then fused with “immortal” myeloma cells (murine in the example) so as to give rise to hybridomas. Using the heterogeneous mixture of cells thus obtained, a selection of the cells capable of producing a particular antibody and of multiplying indefinitely is then carried out. Each hybridoma is multiplied in the form of a clone, each resulting in the production of a monoclonal antibody of which the recognition properties with respect to the protein may be tested, for example, by ELISA, by one-dimensional or two-dimensional Western blotting, by immunofluorescence, or by means of a biosensor. The monoclonal antibodies thus selected are subsequently purified, especially according to the affinity chromatography technique described above.


The monoclonal antibodies may also be recombinant antibodies obtained by genetic engineering, using techniques well known to those skilled in the art.


The term “antibody analogs” is intended to mean biological and/or chemical compounds which have the same binding capacities as the antibodies or antibody fragments or similar binding capacities. In particular, the antibody analogs include small proteins which, like antibodies, are capable of binding to a biological target thus making it possible to detect it, to capture it or quite simply to target it within an organism or within a biological sample. The fields of application of these antibody analogs are virtually as vast as those of antibodies. By way of example, mention may be made of the Nanofitins™, which are small proteins sold by the company Affilogic.


The binding partners specific for the desired protein can be used as a capture reagent, as a detection reagent or as capture and detection reagents.


The visualization of the immunological reactions, i.e. the protein/binding partner binding, can be carried out by any means of detection, via labeling, of the binding partner.


The term “labeling” is intended to mean the binding of a label reagent capable of generating a detectable signal, i.e. a compound, a substance or a particle which can be detected by visual, fluorescent or instrumental means.


A nonlimiting list of these label reagents consists of:

    • metal or alloy particles, such as colloidal gold particles,
    • polymer particles, such as colored latex particles,
    • magnetic particles,
    • fluorescent molecules,
    • chemoluminescent molecules.


By way of example of immunological tests as defined above, mention may be made of “sandwich” and “competition” methods.





FIGURES

The majority of the figures illustrate the validation of the results by a quantitative ELISA assay carried out on individual samples taken from patients during the acute febrile phase of the disease, before defervescence. The patients having remained with conventional dengue are denoted DF and the patients having then developed severe dengue are denoted SevD. In all cases, the reading is carried out at an optical density (OD) of 450 nm. The results were obtained on samples with different geographical origins: Columbia and Cambodia. On the graphs obtained (GraphPad Prism software, V4.03), the median calculated is represented by a horizontal line. The box illustrates the values encompassing 50% of the individuals. The maximum and minimum values are also illustrated. The values taken into account correspond to the mean of two independent tests carried out in duplicate.



FIG. 1 illustrates the presence of virus in the fractions purified from plasma from patients but not in the control (C), demonstrated with Western blotting using a monoclonal antibody directed against the viral protein E.



FIG. 2 illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the OLFM4 marker on plasma samples from Colombian patients.



FIG. 3 illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the OLFM4 marker on plasma samples from Cambodian patients.



FIG. 4 illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the PF4 marker on plasma samples from Colombian patients.



FIG. 5 illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the PF4 marker on plasma samples from Cambodian patients.



FIG. 6 illustrates the results obtained for the quantitative assaying by means of an ELISA assay of the A2M marker on plasma samples from Cambodian patients.





EXAMPLE 1: CHARACTERIZATION OF THE SAMPLES

15 Colombian plasma samples positive for dengue were selected, from which 8 originate from patients remaining with conventional dengue without developing severe dengue (patients/samples referred to hereinafter as DF or conventional dengue) and 7 originate from patients having then developed severe dengue (patients/samples referred to hereinafter as SevD or severe dengue). The various plasmas were grouped together, composing respectively a pool of conventional dengue DF plasma and a pool of severe dengue SevD plasma for those having developed severe dengue. All the plasmas were taken during the acute febrile phase of the disease, before the critical phase, from patients having a secondary infection. The serotypes concerned were serotypes 1, 2 and 3. All the patients having developed severe dengue were hospitalized and had signs of hemorrhaging. No comorbidity was reported [5]. All the plasmas were verified as being NS1-positive (Platelia dengue kit, Bio-rad) and the viral load was also verified by Q-RT-PCR with a commercially available kit (PrimerDesign) following supplier instructions: the mean number of viral RNA copies was estimated at 4×106 and 4.1×10′ for the conventional dengue (DF) pools and severe dengue (SevD) pools, respectively. The pools composed correspond to a volume of approximately 2 ml of plasma. Before purification, the plasma mixtures were centrifuged for 5 mins at 1000×g and at 4° C. so as to remove the impurities present in the sample and to obtain clarified samples.


The plasma selection criteria are described in table 1 below. The samples were taken after appearance of symptoms.











TABLE 1





Colombian plasma
DF POOL
SevD POOL


















General
Number
8
7



Age (mean)
26.8
33.7



M/F ratio
6/2
3/4



Day
3.3
2.7



Secondary infection
Yes
Yes



NS1
Positive
Positive


Serotype
DV1
1
0



DV2
1
3



DV3
6
4









These plasma sample pools were then purified to obtain virus-enriched fractions as described below.


EXAMPLE 2: PURIFICATION OF THE SAMPLES

All the steps are carried out at 4° C. The clarified samples are supplemented with 8 ml of cold pH8 PBS (PBS8), then centrifuged for 2 h at 41 000×g in an Optima L90 ultracentrifuge (Beckman). The rotor used is the SW41 rotor (Beckman). After centrifugation, the supernatant is removed and the viral pellet obtained is resuspended in 200 microliters of PBS8 then loaded onto a discontinuous gradient composed of 5 ml of 60% (w/w) sucrose in PBS8 and 5 ml of 20% (w/w) sucrose in PBS8. After renewed centrifugation for 2 h at 41 000×g, a virion-enriched ring located at the interface between the two sucrose solutions is taken off with a pipette, diluted 10 times with PBS8 and finally centrifuged one last time for 2 h at 41 000×g. The pellet obtained is resuspended in 200 microliters of PBS8.


This resuspension is then purified using an insoluble polyelectrolyte, Viraffinity (BioSupportGroup, USA). For this purpose, 200 microliters of an MN buffer (60 mM MES pH 6.5, 150 mM NaCl) are added to the viral suspension along with 100 microliters of Viraffinity. The mixture is incubated for 5 min at room temperature then centrifuged for 10 min at 1000×g, following supplier instructions. The supernatant is removed and the polymer pellet is rinsed 3 times with 200 microliters of MN buffer. The viral proteins are recovered by heating the polymer for 5 min/70° C. in the presence of 50 microliters of a buffer containing SDS (Novex InVitrogen) then centrifugation for 5 min at 1000×g.


The presence of the virus in the final samples was verified by immunoblotting with a monoclonal antibody directed against the envelope protein of the dengue virus (E protein). As illustrated in FIG. 1, strong signals at 60 KDa and 120 KDa, corresponding respectively to the monomeric and dimeric forms of the envelope protein, are specifically detected by the monoclonal antibody in the plasma pools. On the contrary, the envelope protein was not detected on a control corresponding to a pool of healthy (non-dengue) plasma purified in the same way as has been described above.


EXAMPLE 3: IDENTIFICATION OF THE SPECIFIC PROTEINS FOR EACH PLASMA POOL, CONVENTIONAL DENGUE DF AND SEVERE DENGUE SEVD, BY MASS SPECTROMETRY (MS)

Method:


The viral preparations and the control sample obtained according to example 2 are deposited on a non-denaturing polyacrylamide gel and migrated until the proteins penetrate into the gel, in order to desalify the sample. The band containing the proteins is excised manually then washed three times in a buffer containing 50% acetonitrile then finally dried in 100% acetonitrile. The gel is then rehydrated in a 7% H2O2 solution before being washed again. A solution of trypsin diluted in 25 mM NH4HCO3 is then added for hydrolysis at 37° C. overnight. The peptides thus obtained are extracted by 15 minute sequential extractions with 30 microliters of 50% acetonitrile, 30 microliters of 5% formic acid and 30 microliters of 100% acetonitrile. These sequential extractions are mixed, dried under vacuum and resuspended in a solution containing 5% acetonitrile and 0.1% trifluoroacetic acid. After quantification of the samples, a defined quantity of peptides is analyzed by nano liquid chromatography coupled together with mass spectrometry (Ultimate 3000, Dionex and LTQ-Orbitrap VelosPro, Thermo Fisher Scientific). The results are acquired by virtue of the Xcalibur software (Thermo Fisher) and automatically converted by the Mascot Daemon V2.2 software (Matrix Science). Searching is then carried out on the Swissprot and Trembl databases via Mascot 2.2. Each experiment was carried out twice, independently. The proteins were identified by the EDyP Service laboratory (CEA Grenoble, France).


Results:


The viral envelope protein E was repeatedly identified in the samples containing virus. The predominantly identified peptide sequence is GWGNGCGLLFKG. This result confirms the presence of the virus in the purified fraction.


For the proteins of cellular origin, identified by proteomics on purified plasma pools, the results obtained are summarized in tables 2a and 2b. In these tables, only those proteins having a variance of less than 25% for the number of peptides found from one experiment to the other have been considered. Similarly, for the severe dengue sample, a number of peptides of greater than 2 was required. According to these criteria, 189 proteins were finally selected. These proteins are described in tables 2a and 2b below. A ratio of “number of peptides in severe dengue (SevD) sample”/“number of peptides in conventional dengue (DF) sample” (SevD/DF) could be calculated for the majority of these proteins (cf. table 2a). Some proteins were only identified in the SevD sample (cf. table 2b); in this case, the SevD/DF ratio could not be calculated.













TABLE 2a







Mean
Mean





number
number




of
of
SevD/DF


Accession

peptides
peptides
peptides


number
Protein name
DF
SevD
ratio



















P09871
Complement C1s subcomponent (C1
3.5
14.5
4.14



esterase)


P07225
Vitamin K-dependent protein S
1
4
4.00


P01008
Antithrombin-III (ATIII) (Serpin C1)
3
9.5
3.17


Q16610
Extracellular matrix protein 1
1
3
3.00



(Secretory component p85)


P27918
Properdin (Complement factor P)
1.5
4
2.67


B4E1B2
Serotransferrin
11
29
2.64


Q53H26
Transferrin variant (Fragment)
11
29
2.64


B4DPQ0
Complement C1r subcomponent
5
13
2.60


P01019
Angiotensinogen (Serpin A8)
1
2.5
2.50


B4DDU2
Tubulin alpha-ubiquitous chain
2.5
6
2.40


P01779
Ig heavy chain V-III region TUR
2.5
6
2.40


P68366
Tubulin alpha-4A chain (Alpha-
3
6.5
2.17



tubulin 1)


P00734
Prothrombin (Coagulation factor II)
2
4
2.00


P21333
Filamin-A (Actin-binding protein
14.5
28.5
1.97



280)


P00450
Ceruloplasmin (EC 1.16.3.1)
8.5
16.5
1.94



(Ferroxidase)


E7EX29
14-3-3 protein zeta/delta (Fragment)
4.5
8.5
1.89


P63104
14-3-3 protein zeta/delta (KCIP-1)
4.5
8.5
1.89


B4E1D8
cDNA FLJ51597, highly similar to
10.5
19
1.81



C4b-binding protein


P18206
Vinculin (Metavinculin)
5
9
1.80


P12814
Alpha-actinin-1 (Alpha-actinin
6.5
11.5
1.77



cytoskeletal isoform)


O43707
Alpha-actinin-4 (F-actin cross-
6.5
11.5
1.77



linking protein)


P06396
Gelsolin (AGEL) (Actin-
3.5
6
1.71



depolymerizing factor)


P05106
Integrin beta-3 (Platelet membrane
5.5
9
1.64



glycoprotein IIIa) (GPIIIa)


P08514
Integrin alpha-IIb (GPIIb)
12.5
20
1.60



(Platelet membrane glycoprotein IIb)


P01833
Polymeric immunoglobulin receptor
2.5
4
1.60



(PIgR)


Q9Y490
Talin-1
28.5
43
1.51


A2J1N9
Rheumatoid factor RF-ET12 (Fragment)
3
4.5
1.50


Q5NV90
V2-17 protein (Fragment)
3
4.5
1.50


P01023
α-2-macroglobulin (Alpha-2-M)
56.5
83
1.47


A2KBC6
Anti-Factor VIII scFv (Fragment)
6.5
9.5
1.46


P01011
Alpha-1-antichymotrypsin (ACT)
3.5
5
1.43



(Serpin A3)


P00738
Haptoglobin
13.5
19
1.41


P02743
Serum amyloid P-component (SAP)
2.5
3.5
1.40


P01024
C3 and PZP-like α-2-macroglobulin
60.5
84.5
1.40



domain-containing protein 1


A8K008
cDNA FLJ78387
17.5
24
1.37


A2IPI5
HRV Fab 026-VL (Fragment)
3
4
1.33


Q6UX06
Olfactomedin 4 (OLM4) (Antiapoptotic
4.5
6
1.33



protein GW112)


P03952
Plasma kallikrein (EC 3.4.21.34)
1.5
2
1.33



(Fletcher factor)


P08567
Pleckstrin (Platelet 47 kDa protein)
1.5
2
1.33



(p47)


P07996
Thrombospondin-1
19.5
26
1.33


P07437
Tubulin beta chain (Tubulin beta-5
4.5
6
1.33



chain)


A2MYD4
V2-7 protein (Fragment)
3
4
1.33


P00488
Coagulation factor XIII A chain (EC
3.5
4.5
1.29



2.3.2.13)


Q71U36
Tubulin alpha-1A chain (Alpha-
3.5
4.5
1.29



tubulin 3)


P68363
Tubulin alpha-1B chain (Alpha-
3.5
4.5
1.29



tubulin ubiquitous)


Q9BQE3
Tubulin alpha-1C chain (Alpha-
3.5
4.5
1.29



tubulin 6)


Q9H4B7
Tubulin beta-1 chain
3.5
4.5
1.29


Q96K68
cDNA FLJ14473 fis, clone
12
15
1.25



MAMMA1001080


P08107
Heat shock 70 kDa protein 1A/1B
2
2.5
1.25


P34931
Heat shock 70 kDa protein 1-like
2
2.5
1.25


P11142
Heat shock cognate 71 kDa protein
2
2.5
1.25


Q6N089
Putative uncharacterized protein
15
18.5
1.23



DKFZp686P15220


P02675
Fibrinogen beta chain
11
13.5
1.23


P00739
Haptoglobin-related protein
12
14.5
1.21


Q86UX7
Fermitin family homolog 3 (Kindlin-
5
6
1.20



3)


P02751
Fibronectin (FN)
61.5
72.5
1.18


B7ZLE5
FN1 protein
61.5
72.5
1.18


Q6MZM7
Putative uncharacterized protein
61.5
72.5
1.18



DKFZp686O12165


Q68CX6
Putative uncharacterized protein
61.5
72.5
1.18



DKFZp686O13149


P68032
Actin, alpha cardiac muscle 1
8.5
10
1.18



(Alpha-cardiac actin)


Q562R1
Beta-actin-like protein 2 (Kappa-
8.5
10
1.18



actin)


P0C0L4
Complement C4-A (Acidic complement
52
61
1.17



C4)


P0C0L5
Complement C4-B (Basic complement
52
61
1.17



C4)


B0UZ85
Complement component 4B (Childo
53
62
1.17



blood group)


Q6MZQ6
Putative uncharacterized protein
15
17.5
1.17



DKFZp686G11190


P07477
Trypsin-1 (EC 3.4.21.4) (Serine
3
3.5
1.17



protease 1)


Q4TZM4
Hemoglobin beta chain (Fragment)
6.5
7.5
1.15


P04004
Vitronectin (VN) (S-protein) (V75)
6.5
7.5
1.15


B2RUT6
Complement component 4A (Rodgers
53.5
61.5
1.15



blood group)


P01031
Complement C5
3.5
4
1.14


Q9UL78
Myosin-reactive immunoglobulin light
3.5
4
1.14



chain variable region


Q5NV62
V3-4 protein (Fragment)
6
7
1.17


A6H8M8
C4A protein (Complement C4 gamma
53.5
61
1.14



chain)


P02768
Serum albumin
51.5
58.5
1.14


Q7Z351
Putative uncharacterized protein
15
17
1.13



DKFZp686N02209


P02649
Apolipoprotein E (Apo-E)
4
4.5
1.13


P01009
Alpha-1-antitrypsin (Alpha-1
8.5
9.5
1.12



protease inhibitor) (Serpin A1)


Q08380
Galectin-3-binding protein (Basement
12
13
1.08



membrane autoantigen p105)


Q6N092
Putative uncharacterized protein
12
13
1.08



DKFZp686K18196 (Fragment)


P63261
Actin, cytoplasmic 2 (Gamma-actin)
20
21.5
1.08


P02671
Fibrinogen alpha chain
9.5
10
1.05


P60709
Actin, cytoplasmic 1 (Beta-actin)
19.5
20.5
1.05


A2MYE1
A30 (Fragment)
4
4
1.00


Q96SA9
Anti-streptococcal/anti-myosin
3.5
3.5
1.00



immunoglobulin kappa light chain


P06576
ATP synthase subunit beta,
2
2
1.00



mitochondrial (EC 3.6.3.14)


O43866
CD5 antigen-like (CT-2) (IgM-
15
15
1.00



associated peptide)


P12259
Coagulation factor V (Activated
1.5
1.5
1.00



protein C cofactor)


P02747
Complement C1q subcomponent subunit
5.5
5.5
1.00



C


P04196
Histidine-rich glycoprotein (HPRG)
1.5
1.5
1.00


P01892
HLA class I, A-2 alpha chain (MHC
3
3
1.00



class I antigen A*2)


P30447
HLA class I histocompatibility
3
3
1.00



antigen, A-23 alpha chain


F6IQP2
MHC class I antigen (Fragment)
3
3
1.00


F6IR35
MHC class I antigen (Fragment)
3
3
1.00


A2NKM7
NANUC-2 heavy chain (Fragment)
3
3
1.00


P26022
Pentraxin-related protein PTX3
2
2
1.00



(Pentaxin-related protein PTX3)


P02760
Protein AMBP
2.5
2.5
1.00


A2J1M8
Rheumatoid factor RF-IP12
3
3
1.00



(Rheumatoid factor RF-IP13)


A2J1N0
Rheumatoid factor RF-IP14 (Fragment)
6
6
1.00


Q9HCC1
Single chain Fv (Fragment)
7.5
7.5
1.00


H0YLA9
Uncharacterized protein
2
2
1.00


P02774
Vitamin D-binding protein (DBP)
2
2
1.00



(VDB)


P02647
Apolipoprotein A-I (ApoA-I)
11
10.5
0.95



(Apolipoprotein A1)


P68871
Hemoglobin subunit beta (Beta-
8.5
8
0.94



globin)


A2NYQ9
Anti-folate binding protein
7
6.5
0.93



(Fragment)


A2JA14
Anti-mucin1 heavy chain variable
6.5
6
0.92



region (Fragment)


B6EDE2
Epididymis luminal protein 180
6.5
6
0.92



(Fragment)


Q14477
Hbbm fused globin protein (Fragment)
6.5
6
0.92


Q670S4
Hemoglobin Lepore-Baltimore
6.5
6
0.92



(Fragment)


P02042
Hemoglobin subunit delta (Delta-
6.5
6
0.92



globin)


Q9UL90
Myosin-reactive immunoglobulin heavy
6.5
6
0.92



chain variable region


A2NZ55
Variable immnoglobulin anti-
6.5
6
0.92



estradiol heavy chain


Q15485
Ficolin-2 (37 kDa elastin-binding
5.5
5
0.91



protein)


Q9UL88
Myosin-reactive immunoglobulin heavy
5
4.5
0.90



chain variable region


Q6ZVX0
cDNA FLJ41981 fis, clone
9.5
8.5
0.89



SMINT2011888


Q7Z374
Putative uncharacterized protein
9.5
8.5
0.89



DKFZp686C02218


Q6MZX9
Putative uncharacterized protein
9.5
8.5
0.89



DKFZp686M08189


P02679
Fibrinogen gamma chain
12.5
11
0.88


Q6P5S8
antioxidant activity; very-low-
16
14
0.88



density lipoprotein particle



remodeling


P27105
Erythrocyte band 7 integral membrane
4
3.5
0.88



protein (Stomatin)


P69905
Hemoglobin subunit alpha (Alpha-
4
3.5
0.88



globin)


Q6GMX0
Putative uncharacterized protein
16
14
0.88


P02792
Ferritin light chain (Ferritin L
3.5
3
0.86



subunit)


Q9UL71
Myosin-reactive immunoglobulin heavy
7
6
0.86



chain variable region


Q07954
Prolow-density lipoprotein receptor-
7
6
0.86



related protein 1 (LRP-1)


A1A508
PRSS3 protein
3.5
3
0.86


Q6P5S3
Putative uncharacterized protein
7
6
0.86


Q65ZC9
Single-chain Fv (Fragment)
7
6
0.86


Q5CZ94
Putative uncharacterized protein
6.5
5.5
0.85



DKFZp781M0386


P19823
Inter-alpha-trypsin inhibitor heavy
9.5
8
0.84



chain H2 (ITI-HC2)


Q16195
Keratin (Fragment)
9.5
8
0.84


P30464
HLA class I, B-15 alpha chain (MHC
3
2.5
0.83



class I antigen B*15)


P03989
HLA class I, B-27 alpha chain (MHC
3
2.5
0.83



class I antigen B*27)


Q9UL83
Myosin-reactive immunoglobulin light
3
2.5
0.83



chain variable region


P35579
Myosin-9 (Myosin heavy chain 9)
8.5
7
0.82


A0A5E4
Putative uncharacterized protein
8.5
7
0.82


P10909
Clusterin (Apolipoprotein J) (Apo-J)
5.5
4.5
0.82


Q5EFE6
Anti-RhD monoclonal T125 kappa light
16
13
0.81



chain


Q86TT1
Full-length cDNA clone CS0DD006YL02
28
22.5
0.80



of Neuroblastoma


O14791
Apolipoprotein L1 (ApoL-I)
2.5
2
0.80


A2NB45
Cold agglutinin FS-1 L-chain
2.5
2
0.80



(Fragment)


A2NB44
Cold agglutinin FS-2 H-chain
2.5
2
0.80



(Fragment)


B1N7B8
Cryocrystalglobulin CC1 kappa light
2.5
2
0.80



chain variable region


Q86SX2
Full-length cDNA clone CS0DL004YM19
2.5
2
0.80



of B cells


Q7Z3Y6
Rearranged VH4-34 V gene segment
2.5
2
0.80



(Fragment)


A0N5G5
Rheumatoid factor D5 light chain
2.5
2
0.80



(Fragment)


H0YK52
Uncharacterized protein
2.5
2
0.80


Q14624
Inter-alpha-trypsin inhibitor heavy
16
12.5
0.78



chain H4 (ITI-HC4)


P31946
14-3-3 protein beta/alpha (KCIP-1)
4.5
3.5
0.78


P62258
14-3-3 protein epsilon (14-3-3E)
4.5
3.5
0.78


Q04917
14-3-3 protein eta (Protein AS1)
4.5
3.5
0.78


P61981
14-3-3 protein gamma (KCIP-1)
4.5
3.5
0.78


P27348
14-3-3 protein theta (Protein HS1)
4.5
3.5
0.78


A2NUT2
Lambda-chain (AA −20 to 215)
9
7
0.78


P02776
Platelet factor 4 (PF-4)
4
3
0.75


P10720
Platelet factor 4 variant (CXCL4L1)
4
3
0.75


P02746
Complement C1q subcomponent subunit
9.5
7
0.74



B


P48740
Mannan-binding lectin serine
7.5
5.5
0.73



protease 1 (EC 3.4.21.—)


Q96JD0
Amyloid lambda 6 light chain
9
6.5
0.72



variable region SAR (Fragment)


Q6GMV8
Putative uncharacterized protein
9
6.5
0.72


Q8NEJ1
Putative uncharacterized protein
9
6.5
0.72


A0N5G3
Rheumatoid factor G9 light chain
9
6.5
0.72



(Fragment)


P02745
Complement C1q subcomponent subunit
3.5
2.5
0.71



A


P60660
Myosin light polypeptide 6 (MLC-3)
3.5
2.5
0.71


Q6MZU6
Putative uncharacterized protein
17.5
12.5
0.71



DKFZp686C15213


Q6N093
Putative uncharacterized protein
17.5
12.5
0.71



DKFZp686I04196 (Fragment)


P61224
Ras-related protein Rap-1b (GTP-
3
2
0.67



binding protein smg p21B)


P19105
Myosin regulatory light chain 12A
4
2.5
0.63



(MLC-2B)


P05387
60S acidic ribosomal protein P2
2.5
1.5
0.60



(Renal carcinoma antigen NY-REN-44)


P19338
Nucleolin (Protein C23)
10
6
0.60


O75636
Ficolin-3 (Collagen/fibrinogen
3.5
2
0.57



domain-containing lectin 3 p35)


F8VWA4
Uncharacterized protein
3.5
2
0.57


B7Z539
cDNA FLJ56954
8
4.5
0.56


Q96CX2
BTB/POZ domain-containing protein
4.5
2.5
0.56



KCTD12 (Pfetin)


P02730
Band 3 anion transport protein(AE 1)
17.5
9.5
0.54


P23528
Cofilin-1 (p18)
3
1.5
0.50


P06748
Nucleophosmin (NPM)
3
1.5
0.50


Q6N091
Putative uncharacterized protein
7
3.5
0.50



DKFZp686C02220


Q6N041
Putative uncharacterized protein
7
3.5
0.50



DKFZp686O16217 (Fragment)


P11166
Solute carrier family 2, facilitated
2
1
0.50



glucose transporter member 1


P04275
von Willebrand antigen 2
61.5
28.5
0.46


P62805
Histone H4
2.5
1
0.40


Q13201
Multimerin-1 (EMILIN-4)
11
4
0.36


B7Z1F8
cDNA FLJ53025, highly similar to
52
16
0.31



Complement C4-B


P04114
Apolipoprotein B-100 (Apo B-100)
25
7.5
0.30


Q8TCG3
TPMsk3 (Fragment)
5
1.5
0.30


P16452
Erythrocyte membrane protein band
5.5
1
0.18



4.2 (P4.2)


P11277
Spectrin beta chain, erythrocyte
23
3
0.13



(Beta-I spectrin)


P02549
Spectrin alpha chain, erythrocyte
33
1.5
0.05



(Erythroid alpha-spectrin)




















TABLE 2b









Mean





number





of



Accession

peptides



Number
Protein name
SevD




















P00751
Complement factor B (C3/C5
4.5




convertase)



P00752
Complement component C8 alpha chain
2



P00753
Peroxiredoxin-1 (Natural killer
2




cell-enhancing factor A)



P00754
Moesin (Membrane-organizing
2




extension spike protein)



P00755
Chloride intracellular channel
2




protein 1



P00756
Ferritin heavy chain (Ferritin H
2




subunit)










EXAMPLE 4: CONFIRMATION ELISA

Method:


So as to confirm the mass spectrometry results, specific quantitative ELISAs were carried out in duplicate on individual plasmas. The proteins selected and tested, from those identified in tables 2a/2b, are those with a severe dengue (SevD)/conventional dengue (DE) ratio of greater than or equal to 1.33 and less than or equal to 0.75 with a mean number of peptides of greater than 1 for each sample and a potential link to dengue pathogenesis. This first screening made it possible to only assay those proteins most of interest. According to these criteria, the following proteins were selected:

    • ceruloplasmin,
    • protein S,
    • properdin complement factor,
    • antithrombin-III,
    • secretory component p85,
    • complement C1r protein,
    • complement C1s protein,
    • angiotensin,
    • factor II,
    • CFB,
    • anti-factor VIII,
    • serum amyloid P-component,
    • olfactomedin 4 (OLFM4),
    • thrombospondin,
    • platelet factor 4 (PF4),
    • complement C1q protein,
    • moesine, and
    • complement C8 protein.


Multimerin-1, apolipoprotein B-100 and von Willebrand factor were also assayed.


It should be noted that these proteins are predominantly elements of the coagulation pathway or the complement cascade.


These ELISAs were carried out by virtue of commercially available kits (USCN, China), following supplier instructions. Statistical analyses (Mann-Whitney test and ROC/AUC curve) were carried out by means of GraphPad Prism V4.03 software.


Each candidate marker was assayed on individual plasma samples. These samples are plasma samples taken during the acute febrile phase of the disease (viremic phase), these samples either originating from patients having remained with conventional dengue DF, without developing severe dengue, or from patients having developed severe dengue SevD. All the patients had secondary dengue. Only serotypes 1, 2 and 3 were represented (no serotype 4). These samples originated from l'Universidad Industrial de Santander (Bucaramanga, Colombia) [5] or from the Institut Pasteur in Cambodia (Phnom-Penh). The latter were part of a prospective study carried out in agreement with the local ethics committee. The characteristics of the two sampling sources are given in tables 3 and 4 below. The samples were collected after appearance of symptoms.









TABLE 3







Colombian plasma samples











DF (n = 15)
SevD (n = 15)
P value














Mean age +− SD
29.3 +− 0.52
28.3 +− 0.5 
ns


(years)


% male
60%
53%
ns


Sampling (day)
3.06
2.37
ns


Mean weight +−
60.2 +− 8.6 
50.3 +− 15.6
ns


SD (kg)


Total cholesterol
1.44 +− 5.4 
1.29 +− 10.1
0.03 


(g/l)


AST (U/L)
90.2 +− 9.75
142.7 +− 29.8 
0.001


ALT (U/L)
69.7 +− 11.4
90.8 +− 24.5
ns


Positive tourni-
8/15 (60%)
10/15 (66%)
ns


quet test


Viral load
4.05 106 +− 3.5  
4.1 107 +− 5.22 
0.006


(copies/ml)


Serotype
DV2-DV3
DV2-DV3


Secondary dengue
Yes
Yes


Comorbidity
No
No
















TABLE 4







Cambodian plasma samples











DF (n = 23)
SevD (n = 26)
P value














Mean age +− SD
 8.6 +− 0.38
 7.3 +− 0.45
ns


(years)


% male
56%
35.6%
ns


Sampling (day)
2.6
3.2
ns


Mean weight +−
20.48 +− 0.93 
18.86 +− 0.98 
ns


SD (kg)


Total cholesterol
3.32 +− 0.14
2.37 +− 0.12
<0.0001


(mmol/l)


HDL (mmol/l)
0.82 +− 0.07
0.31 +− 0.02
<0.0001


Triglycerides (g/l)
1.37 +− 0.17
2.8 +− 0.2
<0.0001


AST (IU/L)
133.3 +− 23.18
302.5 +− 50.7 
 0.0043


ALT (IU/L)
66.9 +− 13.4
106.3 +− 21.2 
ns


Viral load
7.1 108 +− 6   
2.06 108 +− 2    
ns


(copies/ml)


Positive tourni-
19/23 (82%)
18/26 (82.6%)
ns


quet test


HGB
11.63 +− 0.15 
13.3 +− 0.29
<0.0001


Hematocrit
38.23 +− 0.57 
41.62 +− 0.8 
 0.0013


Hepatomegaly
 8/12 (66%)
16/26 (61.5%)
ns


(ultrasound)


Serotype
DV1
DV1



Secondary dengue
Yes
Yes



Comorbidity
No
No






SD: standard deviation


ns: non-significant p value






Results:


For the majority of the ELISA-assayed markers, no difference in plasma concentration was observed between the DF and SevD plasmas, whether Colombian or Cambodian (p>0.1).


On the other hand, for two markers, the results make it possible to clearly distinguish those patients who then developed severe dengue SevD from those who remained solely with conventional dengue without developing severe dengue. The first marker is OLFM4 (olfactomedin 4).


For the Colombian samples, the plasma concentration of the marker is higher in the SevD samples compared to the DF samples (p=0.07; cf. FIG. 2).


This is confirmed on the Cambodian samples with an extremely significant difference in concentration (p<0.0003) and a median that is more than twice as high for the SevD samples compared to the DF samples (FIG. 3). The AUC is 0.858 (95% CI=0.7307-0.985). The ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 5: for this marker and for a sensitivity close to 95%, a specificity of greater than 72% is reached.












TABLE 5







For a sensitivity of:
The best specificity is:




















OLFM4
100
61




94.4
72.2












    • The second marker is PF4 (platelet factor 4)





For the Colombian samples, a difference in plasma concentration in favor of the DF samples is observed (p<0.001) (FIG. 4). The AUC is 0.88 (95% CI: 0.7305-1).


This is confirmed for the Cambodian samples for which there is a significant difference in plasma concentration in favor of the DF samples (p<0.0001) (FIG. 5). The AUC is 0.94 (95% CI=0.87-1). The ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 6 below: for this marker and for a sensitivity of 95%, a specificity of close to 78% is reached.












TABLE 6







For a sensitivity of:
The best specificity is:




















PF4
100
61




95
77.9










In parallel, another marker, α-2 macroglobulin (A2M) was identified from unpurified Cambodian plasma samples by a SILAC-type differential proteomic method (Stable Isotope Labelling by Aminoacids in cell Culture) [6]. The identification of this third marker is described in the following examples.


EXAMPLE 5: CHARACTERIZATION OF THE SAMPLES

The composition of each plasma pool or group used in this experiment is summarized in table 7. All the Cambodian plasmas selected to compose the pools were taken during the acute febrile phase of the disease, before the critical phase, from patients having a secondary infection. The serotype concerned was serotype 1. All the SevD patients were hospitalized and had signs of hemorrhaging. No comorbidity was reported. All the plasmas were verified as being NS1-positive (Platelia dengue kit, Bio-rad) and the viral load was also verified by Q-RT-PCR with a commercially available kit following supplier instructions. The pools composed correspond to a final volume of approximately 2 ml of plasma. The plasma groups are inactivated beforehand with heat (56° C./20 minutes) then preclarified by centrifugation for 5 mins at 1000×g and at 4° C., so as to remove impurities present in the sample.












TABLE 7







DF (n = 6)
SevD (n = 6)




















Serotype
DV1
DV1



Secondary dengue
YES
YES



Age in years (mean)
6-12 (8.6)
6-8 (7)



Male/female
2/4
2/4



NS1 positive/virus positive
YES/YES
YES/YES



Mean day of sampling
4
3.85



Severity grade
0-1
3-4



Comorbidity
NO
NO










EXAMPLE 6: DIFFERENTIAL PROTEOME ANALYSIS

The method used is a semi-quantitative proteomic method of SILAC type (Stable Isotope Labelling by Aminoacids in cell Culture) [6] developed by Pronota (Ghent, Belgium) using the MASStermind'M platform and carried out on conventional dengue DF or severe dengue SevD plasma groups. Each group is composed of a mixture of 6 samples, as detailed in example 5.


These plasma mixtures have been depleted beforehand in the 14 most abundant plasma proteins by affinity chromatography. The quantity of proteins recovered in the end was obtained by a colorimetric assay based on bicinchoninic acid (BCA assay Thermo Fisher Scientific Inc., USA).


The MASStermind™ study compared each sample to a reference sample which groups together all the samples. This method provides information on the relative levels, and presence or absence, of peptides/proteins in the severe dengue SevD samples compared to the conventional dengue DF samples. The differential analysis is carried out by mixing the samples labelled with different isotopes and by analyzing, by mass spectrometry, each matched peak. The isotope label is introduced by tryptic hydrolysis which incorporates 2 18O atoms (“heavy” labeling) onto the C-terminal arginine of a peptide, which leads to a mass difference of 4 daltons to the same peptide labeled with 16O (“light” labeling). The reference sample is labeled with 16O, whereas the individual samples are labeled with 18O. The MS/MS data are then submitted to the MASCOT software for identification of the peptides and proteins in each sample.


Following MS/MS analysis, more than 250 quantifiable proteins were identifiable, 10 proteins of which had at least 1 peptide found to be differential. For each protein identified, the SevD/DF ratio is calculated as the weighted mean of the coefficients of all the peptides identified for the given protein. Overall, the results showed a high degree of similarity between the two proteomes and only a few proteins were found to be expressed differentially. For three of these ten proteins, the peptides identified are systematically expressed differentially and have a mean SevD/DF ratio which deviates from 1 (see the results given in the following table 8). The three proteins identified are: α-2 macroglobulin (A2M), complement C3f and heparin cofactor 2. These proteins are predominantly elements of the coagulation pathway or the complement cascade.












TABLE 8








95% CI on



Accession

mean of


Proteins
number
SevD/DF ratio
ratios







α-2 macroglobulin
A2MG_HUMAN
0.33
0.25-0.44


Complement C3f
CO3_HUMAN
0.89
0.68-1.17


Heparin cofactor 2
HEP2_HUMAN
2.25
1.71-2.95









EXAMPLE 7: CONFIRMATION ELISA

Method:


So as to confirm the mass spectrometry results, specific quantitative ELISAs were carried out in duplicate on individual plasmas. The proteins assayed are those identified in example 6: α-2 macroglobulin (A2M), complement C3 protein, and heparin cofactor 2.


These ELISAs were carried out by virtue of commercially available kits (USCN, China), following supplier instructions. Statistical analyses (Mann-Whitney test and ROC/AUC curve) were carried out by means of GraphPad Prism V4.03 software.


Each candidate marker was assayed on individual samples. These samples were plasma samples taken from patients during the acute febrile phase of the disease (viremic phase). Clinical follow-up of the patients showed that some finally remained with conventional dengue DF without developing severe dengue, whereas others had developed severe dengue SevD. All the patients had secondary dengue. Only serotype 1 was represented. These samples originated from the Institut Pasteur in Cambodia (Phnom-Penh) and were part of a prospective study carried out in agreement with the local ethics committee. The characteristics of the sampling source are given in table 9.













TABLE 9







DF (n = 23)
SevD (n = 26)
P value



















Mean age +− SD
 8.6 +− 0.38
 7.3 +− 0.45
ns


(years)


% male
56%
35.6%
ns


Sampling (day)
2.6
3.2
ns


Mean weight +−
20.48 +− 0.93 
18.86 +− 0.98 
ns


SD (kg)


Total cholesterol
3.32 +− 0.14
2.37 +− 0.12
<0.0001


(mmol/l)


HDL (mmol/l)
0.82 +− 0.07
0.31 +− 0.02
<0.0001


Triglycerides (g/l)
1.37 +− 0.17
2.8 +− 0.2
<0.0001


AST (IU/L)
133.3 +− 23.18
302.5 +− 50.7 
 0.0043


ALT (IU/L)
66.9 +− 13.4
106.3 +− 21.2 
ns


Viral load
7.1 108 +− 6   
2.06 108 +− 2    
ns


(copies/ml)


Positive tourni-
19/23 (82%)
18/26 (82.6%)
ns


quet test


HGB
11.63 +− 0.15 
13.3 +− 0.29
<0.0001


Hematocrit
38.23 +− 0.57 
41.62 +− 0.8 
 0.0013


Hepatomegaly
 8/12 (66%)
16/26 (61.5%)
ns


(ultrasound)


Serotype
DV1
DV1



Secondary dengue
Yes
Yes



Comorbidity
No
No






HBG: plasma hemoglobin


SD: standard deviation


ns: non-significant p value






Results:


For the majority of the ELISA-assayed markers, (complement C3f and heparin cofactor 2) no difference in plasma concentration was observed between the DF and SevD plasmas (p>0.1).


However, for the Cambodian samples ELISA-assayed for A2M, there was a significant difference in concentration (p<0.0004) and a median approximately twice as high for the DF samples compared to the SevD samples (FIG. 6). The AUC was 0.89 (95% CI=0.75-1).


For A2M, the ROC curve made it possible to determine the best specificity for a sensitivity close to 100%. The results are summarized in table 10: for a sensitivity close to 94%, a specificity of greater than 83% is reached.












TABLE 10







For a sensitivity of:
The best specificity is:




















A2M
100
72




93.8
83.3










LITERATURE REFERENCES



  • 1. S B Halstead. The lancet 2007; 370: 1644-52

  • 2. A S Leong et al. Semin. Diagn. Pathol. 2007; 24(4):227-236

  • 3. K. Clyde et al. J. Virol. 2006; 23: 17418-11431

  • 4. Fields Virology, Fifth edition, Knipe D M ed., LWW

  • 5. Villar-Centeno L A et al. Am. J. Trp. Med. Hyg. 2008; 78: 370-374 6. R. Drissi et al. FEBS J. 2013


Claims
  • 1. A method of determining whether a patient infected with dengue virus will develop severe dengue, comprising: obtaining a blood sample collected from the patient infected with dengue virus;measuring the expression level of olfactomedin 4 from the blood sample; andcomparing the expression level of olfactomedin 4 to a reference value obtained from individuals having dengue virus infections without developing severe dengue,wherein, if the expression level of olfactomedin 4 is greater that the reference value, it is determined that the patient will develop severe dengue.
  • 2. The method of claim 1, further comprising detecting dengue virus NS1 protein in the blood sample.
  • 3. The method of claim 1, further comprising measuring the expression level of any of platelet factor 4 and α2-macroglobulin from the blood sample.
  • 4. The method of claim 1, further comprising measuring the expression levels of platelet factor 4 and α2-macroglobulin from the blood sample.
  • 5. The method of claim 1, wherein measuring the expression level of olfactomedin 4 comprises contacting the blood sample with a binding partner for olfactomedin 4 and detecting binding of the binding partner to olfactomedin 4.
  • 6. The method of claim 1, further comprising at least one of: detecting dengue virus NS1 protein in the blood sample;measuring the expression level of platelet factor 4 from the blood sample; ormeasuring the expression level of α2-macroglobulin from the blood sample.
  • 7. The method of claim 6, wherein: measuring the expression level of olfactomedin 4 comprises contacting the blood sample with a binding partner for olfactomedin 4 and detecting binding of the binding partner to olfactomedin 4; anddetecting dengue virus NS1 protein comprises contacting the blood sample with a binding partner for dengue virus NS1 protein and detecting binding of the binding partner to dengue virus NS1 protein;measuring the expression level of platelet factor 4 comprises contacting the blood sample with a binding partner for platelet factor 4 and detecting binding of the binding partner to platelet factor 4; and/ormeasuring the expression level of α2-macroglobulin comprises contacting the blood sample with a binding partner for α2-macroglobulin and detecting binding of the binding partner to α2-macroglobulin.
Priority Claims (1)
Number Date Country Kind
14 51272 Feb 2014 FR national
PCT Information
Filing Document Filing Date Country Kind
PCT/FR2015/050353 2/12/2015 WO 00
Publishing Document Publishing Date Country Kind
WO2015/124851 8/27/2015 WO A
US Referenced Citations (1)
Number Name Date Kind
20100068147 Hibberd et al. Mar 2010 A1
Foreign Referenced Citations (2)
Number Date Country
2013110894 Aug 2013 WO
2013148335 Oct 2013 WO
Non-Patent Literature Citations (15)
Entry
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Related Publications (1)
Number Date Country
20160363590 A1 Dec 2016 US